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| Status |
Public on Jan 11, 2024 |
| Title |
Spleen, PBS treated, replicate 2 |
| Sample type |
SRA |
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| Source name |
Spleen
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| Organism |
Salmo salar |
| Characteristics |
tissue: Spleen
treatment: PBS treatment
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| Treatment protocol |
Twenty fish were anaesthetized using 2-phenoxyethanol (2.5ml in 10L water/ 0.0025% v/v) and given an intraperitoneal injection of either PBS (0.5 mL) (n=10), or the pathogenic Hooke strain of A. salmonicida (2 × 105 colony-forming units / mL in PBS; 0.5 mL/fish) (n=10). Sampling was performed 24 h post-injection. The fish were killed using a Schedule 1 method following overdose anaesthetization using 2-phenoxyethanol (0.1% v/v) and destruction of the brain. Fish were immediately sampled, with spleen samples (approx. 100mg) flash frozen on dry ice before storage at -80°C prior to snRNA-Seq library construction
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| Extracted molecule |
nuclear RNA |
| Extraction protocol |
Approximately 45mg of each frozen spleen sample was placed in a 6-well tissue culture plate (Stem Cell Technologies) with 1 ml TST (2 mL of 2X ST buffer + 120 µL of 1% Tween-20 + 20 µL of 2% BSA brought up to 4 ml with nuclease-free water). The tissue was minced using Noyes Spring Scissors for 10 min on ice. The resulting homogenate was filtered through a 40 µm Falcon cell strainer, and a further 1 mL of TST was added to wash the well and filter. The volume was brought up to 5 mL using 3 mL of 1X ST buffer (diluted from 2xST buffer [292 µl of 146 mM NaCl, 100 µl of 10 mM Tris-HCl pH 7.5, 10 µl of 1 mM CaCl2, 210 µl of 21 mM MgCl2, brought up to 10ml with nuclease-free water]). The sample was centrifuged at 4°C for 5 min at 500g before the resulting pellet was re-suspended in 1 ml 1X ST buffer and the recovered nuclei were filtered through a 40 µm and a 20 µm Falcon cell strainer, Hoechst stained, visually inspected under a fluorescent microscope, and counted using a Bio-Rad TC20.
Spleen nuclei were processed through the 10X Chromium Single Cell Platform using the ChromiumTM Single Cell 30 Library and Gel Bead Kit v3.1 and ChromiumTM Single Cell A Chip Kit (both 10X Genomics) as per the manufacturer’s protocol. For each sample, the nuclei were loaded into a channel of a Chromium 3’ Chip and partitioned into droplets using the Chromium controller before the captured RNA for each nucleus was barcoded and reverse transcribed. The resulting cDNA was PCR amplified for 14 cycles, fragmented, and size selected before Illumina sequencing adaptor and sample indexes were attached.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10X Chromium, 3’ v3.1 chemistry
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| Data processing |
Mapping of reads to the genome, assignment of reads to cellular barcodes, and collapsing of unique molecular identifiers (UMIs) was performed with StarSolo v2.7.8a. The genome index was generated with standard settings and “sjdbOverhang” set to 149. The reads were then mapped with the “STAR” command and following settings: "soloCBstart" = 1, "soloCBlen" = 16, "soloUMIstart" = 17, "soloUMIlen" = 12, "soloBarcodeReadLength" = 0, "soloUMIfiltering" = MultiGeneUMI_CR, "soloCellFilter" = EmptyDrops_CR, "outFilterMatchNmin" = 66, "outFilterScoreMin" = 30, "outFilterMatchNminOverLread" = 0.4, "outFilterScoreMinOverLread" = 0.4, "soloMultiMappers" = EM, "limitOutSJcollapsed" = 2000000, "soloCellReadStats" = Standard.
Assembly: Salmo Salar Ensembl 106 genome
Supplementary files format and content: Tab separated value (tsv) and matrix (mtx) format. Contains STARsolo raw and filtered gene names (features.tsv), cell barcodes (barcodes.tsv), UMI count (matrix.mtx) and the sum of STARsolo unique+multi-gene UMI count (UniqueAndMult-EM.mtx)
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| Submission date |
Jan 09, 2024 |
| Last update date |
Jan 11, 2024 |
| Contact name |
Jianxuan Sun |
| E-mail(s) |
J.Sun-52@sms.ed.ac.uk
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| Organization name |
Roslin Institute
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| Lab |
Macqueen lab
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| Street address |
The University of Edinburgh, Easter Bush Campus
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| City |
Edinburgh |
| State/province |
Midlothian |
| ZIP/Postal code |
EH25 9RG |
| Country |
United Kingdom |
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| Platform ID |
GPL24981 |
| Series (1) |
| GSE252828 |
Cell atlas of the Atlantic salmon spleen reveals immune cell heterogeneity and cell-specific responses to bacterial infection |
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| Relations |
| BioSample |
SAMN39326319 |
| SRA |
SRX23147653 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8008590_pbs6_filtered_barcodes.tsv.gz |
89.3 Kb |
(ftp)(http) |
TSV |
| GSM8008590_pbs6_filtered_features.tsv.gz |
301.1 Kb |
(ftp)(http) |
TSV |
| GSM8008590_pbs6_filtered_matrix.mtx.gz |
76.0 Mb |
(ftp)(http) |
MTX |
| GSM8008590_pbs6_raw_UniqueAndMult-EM.mtx.gz |
164.2 Mb |
(ftp)(http) |
MTX |
| GSM8008590_pbs6_raw_barcodes.tsv.gz |
17.5 Mb |
(ftp)(http) |
TSV |
| GSM8008590_pbs6_raw_features.tsv.gz |
301.1 Kb |
(ftp)(http) |
TSV |
| GSM8008590_pbs6_raw_matrix.mtx.gz |
125.2 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
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